Effects of Hevea brasiliensis Intercropping on the Volatiles of Pandanus amaryllifolius Leaves

Pandanus amaryllifolius Roxb. is a special tropical spice crop resource with broad development prospects. It is widely cultivated under a Hevea brasiliensis (Willd. ex A. Juss.) Muell. Arg. canopy to improve the comprehensive benefits to Hevea brasiliensis plantations in Hainan Provence, China. However, the effects of intercropping with Hevea brasiliensis on the component number and relative contents of volatile substances in different categories in the Pandanus amaryllifolius leaves are still unknown. Therefore, a Hevea brasiliensis and Pandanus amaryllifolius intercropping experiment was set up to clarify the differences between several cultivated patterns on volatile substances in the Pandanus amaryllifolius leaves, and the key regulatory factors of volatile substances. The results showed that the soil pH was significantly decreased, while soil bulk density, alkali-hydrolyzable nitrogen and available phosphorus contents were significantly increased under the intercropping pattern. The component numbers of esters in volatile substances were increased by 6.20%, while the component numbers of ketones were decreased by 4.26% under the intercropping pattern. Compared with the Pandanus amaryllifolius monoculture, the relative contents of pyrroles, esters and furanones were significantly increased by 8.83%, 2.30% and 8.27%, respectively, while the relative contents of ketones, furans and hydrocarbons were decreased by 1.01%, 10.55% and 9.16% under the intercropping pattern, respectively. The relative contents of pyrroles, esters, furanones, ketones, furans and hydrocarbons were associated with changes in soil pH, soil available phosphorus content and air temperature. The results indicated that the reduction in soil pH and enhancement in soil-available phosphorus may be the main reasons for promoting the relative content of pyrroles and reducing the relative content of hydrocarbons under an intercropping pattern. Overall, Hevea brasiliensis intercropping with Pandanus amaryllifolius could not only improve soil properties, but also significantly increase the relative contents of the main volatile substances in Pandanus amaryllifolius leaves, which could provide a theoretical basis for the application and promotion of high-quality production patterns of Pandanus amaryllifolius.


Introduction
Pandanus amarylifolius Roxb. is a tropical perennial herb spice plant, which is native to the Maluku Islands of Indonesia, and widely distributed in South and Southeast Asian countries, such as India, Thailand and Malaysia. As the "Oriental Vanilla" with The experimental region belonged to the tropical monsoon climate. The annual mean temperature, precipitation and sunshine hours were 22 • C, 2100-2200 mm and 1750-2650 h, respectively. The soil was tidal sand-mud (US Soil Taxonomy classification). The soil pH and organic matter content were 6.00 and 20.04 g·kg −1 , respectively.

Experiment Design
Twenty 0.5 m × 1 m experimental plots were established with a randomized block design in this experiment. The blocks were arranged in ten rows with a 1 m buffer between each plot. Each block had two plots, and one plot was set for each planting pattern: Pandanus amaryllifolius monoculture (M), Hevea brasiliensis and Pandanus amaryllifolius intercropping (I). The plant spacing of different crops was 50 cm in each plot. Each treatment was replicated ten times. During the experiment, the management (e.g., fertilization, irrigation, shading, etc.) of all plots was consistent.

Determination of Plant Samples Sample Collection and Pretreatment
The Pandanus amaryllifolius leaves were immediately measured when they were returned to the laboratory. Firstly, the dirt and dust on the leaf surface were removed using gauze and distilled water. Secondly, the middle part of leaf was selected by manually shearing with scissors, in order to avoid the loss of volatile substances caused by the temperature increase during mechanical crushing. Thirdly, 5.0 g of broken fresh leaf were placed into a 50 mL centrifuge tube, and then ultrasonically extracted for 1 h under the conditions of 400 W, 50 • C and 40 KHz. After, 15 mL of absolute ethanol were added into the 50 mL centrifuge tube. Fourthly, the upper liquid was transferred to another 50 mL centrifuge tube via a 0.22 µm organic phase needle filter (nylon). Fifthly, 5 g of anhydrous sodium sulfate were added into the extracted solution samples to remove the water in the liquid. The supernatant was used to detect volatile substances of Pandanus amaryllifolius.

Qualitative Analysis
Qualitative analysis of volatile substances in the Pandanus amaryllifolius leaves was implemented by gas chromatography and mass spectrometry (GC-MS). The results of GC-MS analysis were retrieved from the NIST 2017 spectral library and compared for qualitative analysis: The Piano N-Paraffins Mix (C7~C40, Shanghai Amphora Experimental Technology Co., Ltd., Shanghai, China) was used to conduct GC-MS analysis with the same procedure, and calculate the retention index (RI) of each volatile component according to the linear equation using its retention time [25]. The actually measured RI was compared with that in the literature for qualitative analysis.

Quantitative Analysis
Accurately absorbed 2-acetyl-1-pyrroline (2AP) standard (purity 95%, Toronto Research Chemicals, Canada) and methanol (chromatographic purity) were configured according to 50, 100, 150, 300 and 500 µg mL −1 solution concentration gradients, separately. The standard gradient concentration was injected for GC-MS from low to high, according to the chromatographic conditions of the sample; each concentration was injected three times. The gradient concentration of each component was taken as the abscissa, and the average value of the three measured peak areas was taken as the ordinate, in order to obtain the quantitative linear relationship of each component. The content of 2AP was calculated according to the linear equation using the peak area, and then other volatile substances were semi-quantitated according to the content of 2AP [26]. The calculation formula was Xi = (Ai/As) × Cs (Xi was the content of the substance to be tested; Ai was the peak area of the substance to be tested; As was the peak area of 2AP in standard solution; Cs was the content of 2AP in the sample).

Determination of Soil Samples Sample Collection
Soil samples were collected in October 2021. Three initial soil samples (0-20 cm) were randomly collected from each plot by a 5 cm diameter soil auger, and then mixed as one soil sample for each plot. Plant roots and other visible foreign bodies were removed from soil samples via sieving (<2 mm, <0.20 mm). The soil samples were brought back to analyze soil physico-chemical properties after air drying.

Sample Analysis
Soil pH was measured using a pH/conductivity meter (FE28, China; the soil:water ratio was 1:2.5). After weighing the fresh weight, the soil samples were oven-dried at 105 • C for 24 h, and weighed again to calculate the soil moisture (SM). Soil organic matter (SOM) was determined with a total organic carbon analyzer (Multi N/C 3100, Jena, Germany), and bulk density (SBD, g cm −3 ) was measured as the ratio of soil dry weight and soil volume. The soil alkali-hydrolyzed nitrogen (SAN) was determined using the alkaline hydrolysis diffusion method. Soil available phosphorus (soil Olsen-P, SOP) was assessed using Bray's method (UV2310 II, Shanghai, China). Soil available potassium (SAK) was determined using flame photometry (6400A, Changsha, China).

Determination of Ecosystem Environment
The soil and air temperature (ST and AT, respectively) were measured with a thermocouple probe that connected to the portable soil carbon dioxide flux measurement system (Li-8100, Li-Cor, Inc., Lincoln, NE, USA). The AT was measured at the canopy of Pandanus amaryllifolius (50 cm), and the ST was measured at 20 cm, where Pandanus amaryllifolius roots were widely distributed at this depth. The ST and AT were measured in December 2020, March 2021, June 2021 and September 2021 (once every season). The annual average of AT and ST were used to represent the ecosystem micro-environment indicators for the experimental site.

Statistical Analysis
The t-test was used to determine the differences in experimental indicator and volatile substances (i.e., soil physico-chemical properties, micro-environment indicators, the component numbers and relative contents of volatile categories) between the intercropping and monoculture patterns. Partial least squares discriminant analysis (PLSDA) was used to distinguish the overall difference in volatile substances' relative content between the monoculture and intercropping pattern. The network interaction analysis of soil properties and volatile substances were used to measure the correlation between component numbers or relative contents of volatile substance in different categories and soil properties, as well as the correlation between component numbers or relative contents of volatile substance in different categories. Redundancy analysis (RDA) of environmental factors and relative contents of volatile substance categories was conducted, which was assessed from 999 iterations based on Monte Carlo permutations. Data analyses were performed using SPSS 23.0, SAS V8 and Canaco 5.0. The graphs were plotted using Origin 2021b and Cytoscape V3.8.2.

Effects of Intercropping Pattern on Soil Physico-Chemical Properties and Ecosystem Micro-Environment
The t-test was used to explicate differences in soil physico-chemical properties among different cultivation patterns. Compared with Pandanus amaryllifolius monoculture, the soil pH in the intercropping pattern was significantly decreased by 1.00 (p < 0.05), while SBD, SAN and SOP were significantly increased by 0.24 g cm −3 , 31.01 mg kg −1 and 10.49 mg kg −1 , respectively (p < 0.05). There was no difference between the intercropping and Pandanus amaryllifolius monoculture patterns on SM, SOM and SAK (Table 1). However, compared with Pandanus amaryllifolius monoculture, AT tended to increase by 1.02 • C under the intercropping pattern (p < 0.1). Soil is the basis for crop growth. Improvements in soil micro-environment indices and physico-chemical properties have positive effects on promoting crop physiological activity and increasing productivity [27]. There is a significant impact on the soil properties of tropical perennial plantations under changes in cultivation patterns, especially the intercropping pattern. Previous studies showed that the intercropping pattern could improve the contents of SAP, SAN, SAK and SOM, and promote benign interactions between crops [28,29]. Compared with the Pandanus amaryllifolius monoculture, the intercropping significantly increased the contents of SAN and SOP in this study (Table 1), which is consistent with the results of previous studies [28,29]. Furthermore, the intercropping pattern in the current study significantly improved SBD, which may be related to the root characteristics and activities of the two crops [30]. An increase in SBD usually implies a decrease in soil organic matter content, but there was no significant difference between the SOM of the intercropped and monoculture cultivations in this study (Table 1). The component numbers of Pandanus amaryllifolius volatile substances in different categories under monoculture and intercropping patterns were measured via GC-MS. There were 68 main volatile components in Pandanus amaryllifolius leaves, which were divided into 10 categories, including alcohols, pyrroles, ketones, esters, aldehydes and ketones, furans, furanones, acids, hydrocarbons and phenols (Table A1). The results of t-tests showed that compared with Pandanus amaryllifolius monoculture, the component number of esters significantly increased by 6.20%, while the component number of ketones significantly decreased by 4.26% (p < 0.05, Figure 1a). Compared with the Pandanus amaryllifolius monoculture, the relative contents of pyrroles, esters and furanones significantly increased by 8.83%, 2.30% and 8.27%, respectively (p < 0.05), while the relative contents of ketones, furans and hydrocarbons significantly decreased by 1.01%, 10.55% and 9.16% (p < 0.05), respectively, under the intercropping pattern. There were no differences found between monoculture and intercropping in the relative contents of alcohols, acids, aldehydes, ketones and phenols in Pandanus amaryllifolius leaves (Figure 1b).

Comparison of Pandanus amaryllifolius Volatile Substances under Different Cultivation Patterns
The component numbers of Pandanus amaryllifolius volatile substances in different categories under monoculture and intercropping patterns were measured via GC-MS. There were 68 main volatile components in Pandanus amaryllifolius leaves, which were divided into 10 categories, including alcohols, pyrroles, ketones, esters, aldehydes and ketones, furans, furanones, acids, hydrocarbons and phenols (Table A1). The results of ttests showed that compared with Pandanus amaryllifolius monoculture, the component number of esters significantly increased by 6.20%, while the component number of ketones significantly decreased by 4.26% (p < 0.05, Figure 1a). Compared with the Pandanus amaryllifolius monoculture, the relative contents of pyrroles, esters and furanones significantly increased by 8.83%, 2.30% and 8.27%, respectively (p < 0.05), while the relative contents of ketones, furans and hydrocarbons significantly decreased by 1.01%, 10.55% and 9.16% (p < 0.05), respectively, under the intercropping pattern. There were no differences found between monoculture and intercropping in the relative contents of alcohols, acids, aldehydes, ketones and phenols in Pandanus amaryllifolius leaves (Figure 1b). The Q 2 of PLSDA analysis on the relative contents of volatile substances in different categories under monoculture and intercropping pattern was 0.95, indicating that the model prediction results of this experiment complied with the requirements. There was a significant difference in the relative contents of volatile substances under the Pandanus amaryllifolius monoculture and intercropping patterns in this study (Figure 2), indicating that the different cultivation patterns significantly affected the synthesis and accumulation of volatile substances in Pandanus amaryllifolius leaves. The Q 2 of PLSDA analysis on the relative contents of volatile substances in different categories under monoculture and intercropping pattern was 0.95, indicating that the model prediction results of this experiment complied with the requirements. There was a significant difference in the relative contents of volatile substances under the Pandanus amaryllifolius monoculture and intercropping patterns in this study (Figure 2), indicating that the different cultivation patterns significantly affected the synthesis and accumulation of volatile substances in Pandanus amaryllifolius leaves.

Co-Occurrence Network Analysis between Soil Properties and Volatile Substances of Pandanus amaryllifolius
Co-occurrence network analysis was used to determine the co-occurrence patterns of soil properties, component numbers and relative contents of Pandanus amaryllifolius volatile substances in different categories, based on strong and significant correlations (Figure 3). Overall, different cultivation patterns showed a remarkable effect on the association networks of soil properties and component numbers of volatile substances in different categories. The values of average path length (APL), average connectivity (avgK), average clustering coefficient (avgCC) and graph density in these empirical networks of component numbers of volatile substances in different categories were 1.86, 0.58, 2.46 and 0.21, respectively ( Table 2). More negative co-occurrence relationships were shown between the component numbers of volatile substances in different categories, while more positive co-occurrence relationships were shown from soil properties to the component numbers of volatile substances in different categories in the network graph (Figure 3a). The values of average path length (APL), average connectivity (avgK), average clustering coefficient (avgCC) and graph density in these empirical networks of relative contents of Pandanus amaryllifolius volatile substances in different categories were 2.11, 0.71, 5.20 and 0.37, respectively ( Table 2). More negative co-occurrence relationships were shown between relative contents of Pandanus amaryllifolius volatile substances in different categories, while more positive co-occurrence relationships were shown from soil properties to relative contents of Pandanus amaryllifolius volatile substances in different categories, which indicated that the improvements in soil properties were conducive to increasing component numbers and relative contents of volatile substances in different categories.

Co-occurrence Network Analysis Between Soil Properties of Pandanus amaryllifolius
Co-occurrence network analysis was used to determine the soil properties, component numbers and relative contents of Pa tile substances in different categories, based on strong and signi 3).  The soil pH, among the soil properties, was consistently related to changes in the component numbers of esters, ketones and furanones (Figure 4a and Table 3). Soil pH, SOP and AT were associated with changes in the relative contents of pyrroles, esters, furanones, ketones, furans and hydrocarbons (Figure 4b and Table 3). For the pyrroles, hydrocarbons and alcohols, which affect the key aroma components of the quality of Pandanus amaryllifolius, there was a significant positive correlation between AT, SOP and the relative contents of pyrroles, hydrocarbons and alcohols, while there was an opposite effect on the correlations between pH and relative content of pyrroles, hydrocarbons and alcohols.

Co-occurrence Network Analysis Between Soil Properties and Volatile Substances of Pandanus amaryllifolius
Co-occurrence network analysis was used to determine the co-occurrence patterns of soil properties, component numbers and relative contents of Pandanus amaryllifolius volatile substances in different categories, based on strong and significant correlations ( Figure  3).  Table 2). More negative co-occurrence relationships were shown between the component numbers of volatile substances in different categories, while more positive co-occurrence relationships were shown from soil properties to the component numbers   The soil pH, among the soil properties, was consistently related to changes in the component numbers of esters, ketones and furanones (Figure 4a and Table 3). Soil pH, SOP and AT were associated with changes in the relative contents of pyrroles, esters, furanones, ketones, furans and hydrocarbons (Figure 4b and Table 3). For the pyrroles, hydrocarbons and alcohols, which affect the key aroma components of the quality of Pandanus amaryllifolius, there was a significant positive correlation between AT, SOP and the relative contents of pyrroles, hydrocarbons and alcohols, while there was an opposite effect on the correlations between pH and relative content of pyrroles, hydrocarbons and alcohols.

Key Regulated Factors of Intercropping Pattern on the Component Numbers of Pandanus amaryllifolius Volatile Substances in Different Categories
Pandanus amaryllifolius is a perennial herb spice crop, which is mainly used for its leaves. The main volatile compounds affecting the quality of Pandanus amaryllifolius are alcohols, pyrroles and hydrocarbons, according to the analysis of its leaf compounds from a previous study [31]. In particular, 2AP is the most important substance in pyrroles, which is also the most important volatile aroma substance of Pandanus amaryllifolius, Thai fragrant rice and other crops [32]. It is generally believed that soil properties and planting patterns are the two main factors that affect crop quality [33]. On the one hand, the lack of some soil elements is a key factor limiting the synthesis of certain substances [34], while some excessive soil elements also have a negative feedback effect on the synthesis of certain substances [35]. Improvements in soil nutrients could promote crop productivity and quality by increasing the synthetic substrates of crops [36]. On the other hand, the increased crop species richness could promote the growth and quality of crops by improving soil biological activity, and accelerating the adaptability of crops to the environment under the intercropping pattern [21,37]. Furthermore, there is a competitive relationship between various crops on soil nutrients, which can change the physiological metabolism process of crops, and have a greater impact on crop quality by increasing parts of compound synthesis paths under the intercropping pattern [38]. Therefore, the numbers of compound types in ketones and furanones were significantly and negatively correlated with SAN, SOP and SBD, while there was a positive correlation between esters and the above soil property indexes under the intercropping pattern (Figure 3b) in the current study; these findings indicate that the increased contents of SBD, SAN and SOP under the intercropping pattern may be the main reason for the decrease in ketone and furanone types, and the increase in ester types.

Key Regulated Factors of Intercropping Pattern on the Relative Contents of Pandanus amaryllifolius Volatile Substances in Different Categories
The volatile substances of spice crops are not only determined by gene structure, but also are affected by the growth environment and cultivation methods. Firstly, previous studies proved that the aromatic substance of spearmint increased when the climate and altitude were both appropriate [39]. Compared with the Pandanus amaryllifolius monoculture, air temperatures under the Hevea brasiliensis and Pandanus amaryllifolius intercropping ecosystem showed an upward trend in the current study (Table 1). There was a positive correlation between pyrrole substances' relative contents in Pandanus amaryllifolius leaves and air temperature (Figure 3b), indicating that the increase in air temperature after intercropping may be one of the main factors to improve the physiological activity of the leaves of Pandanus amaryllifolius, and accelerate the synthesis rate of pyrrole substances. Secondly, research shows that Rosa rugosa and Elymus dahurica strengthen the absorption of soil nutrients by increasing the number of root tillers after intercropping with other crops, which has played a potential role in promoting their aromatic components content and productivity [36,40]. The increased contents of SAN and SOP, as well as the positive correlation between SAK, SOP and pyrrole content (Figures 3b and 4b), indicate that the increase in soil nutrients may be another main reason for the accelerated accumulation of pyrrole substances in the current study. Thirdly, the positive correlation between the hydrocarbon content and pH in this study shows that an acidic soil environment after intercropping may be unfavorable for the synthesis and accumulation of hydrocarbons in Pandanus amaryllifolius leaves. Moreover, the negative correlation between hydrocarbon content and SAN and SOP content indicates that an increase in soil nutrients under an intercropping pattern may have a significant negative feedback effect on hydrocarbon synthesis. Overall, the increase in soil nutrient content and the change in ecosystem microenvironment under an intercropping pattern are potential factors affecting the content and quality of volatile substances in Pandanus amaryllifolius leaves. The results of this study provide a theoretical basis for the application and promotion of the Pandanus amaryllifolius and Hevea brasiliensis incropping productive model, by clarifying the impact of environmental factors on the main qualities of Pandanus amaryllifolius under an intercropping pattern.

Conclusions
The volatile substances of Pandanus amaryllifolius leaves significantly changed after intercropping with Hevea brasiliensis. The intercropping pattern decreased component numbers of ketones and furanones, while it had the opposite effect on esters. The relative contents of ketones, furans and hydrocarbons decreased, but the contents of pyrroles, esters and furanones significantly increased under the intercropping pattern. The increased contents of SBD, SOP and SAN, and the decreased pH under the intercropping pattern,

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Appendix A  Figure A1. Effects of monoculture and intercropping patterns on the stomatal conducta photosynthesis (b), transpiration (c), and water use efficiency (d) of Hevea brasiliensis. n Hevea brasiliensis monoculture, I: Pandanus amaryllifolius and Hevea brasiliensis intercro Different letters represent significant differences at p < 0.05.